407. A Novel Rabbit Antibody-Derived, Anti-CD123 LV/CAR Construct for AML Immunotherapy

Abstract

Chimeric antigen receptors (CARs) have emerged in the immunotherapy field as an exciting new option for cancer treatment, with clinical trials of CD19-directed CARs having already demonstrated long-lasting responses in patients with ALL and CLL. As a hematological malignancy, acute myeloid leukemia (AML) may be another viable target for CAR-mediated therapy. Furthermore, with a 5-year survival rate of just 5.5% for patients over 65, new treatments for AML are very much needed.CD123 (the IL-3 receptor α-chain) is upregulated on AML blasts/stem cells and plays a role in proliferation and apoptotic resistance. This antigen demonstrates much lower expression levels on normal hematopoietic cells, where its expression is restricted to the myeloid progenitor subpopulation. Previous attempts to target CD123 in AML through several forms of immunotherapy have had variable success. Specifically, results of previous CD123 murine antibody-derived CARs have been mixed, with some results showing CAR-mediated eradication of normal myelopoiesis via targeting of HSCs with low CD123 expression.We have developed a CD123 CAR, derived from a novel rabbit anti-CD123 mAb that we generated. Rabbit antibodies are reported to have a broader avidity and higher range of affinities than mouse mAbs; this may lead to a CAR with a unique binding profile. We will determine whether such a rabbit-derived CD123 CAR will lead to more specific binding, resulting in optimized killing of AML cells, while minimizing cytotoxic effects on HSCs.To generate our CAR, human CD123 was purified as a GST-tagged protein and used for immunization of rabbits. Hybridoma cell lines were developed from the spleen cells of rabbits with positive immune responses, and novel antibodies were purified and screened for specificity to CD123 using a combination of ELISA, flow cytometry, and ADCC. A candidate antibody was selected, and the VL and VH chains were subcloned, sequenced, and assembled into an scFv. A second generation CAR was then designed that includes a CD8 hinge and transmembrane region, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. This construct was then subcloned into a lentiviral backbone to facilitate expression in immune effector cells.Our CD123 CAR has been transduced into primary T cells and the NK-92 cell line for in vitro testing. Flow cytometry demonstrates that our CARs are expressed at the cell surface. Furthermore, the expression of the CD123 CAR has been shown to be stable in the NK-92 cell line. Cytotoxicity assays are being performed in vitro in order to confirm binding specificity and cytotoxic potential of the CD123 CARs in both NK-92 and T cells. Future work will compare CAR T and CAR NK killing in vivo using NSG mouse models of AML.AML may be an ideal target for CAR therapy, and we will exploit our novel CD123 CARs as therapeutic entities. We will examine whether the use of a novel rabbit anti-CD123 scFv in our LV/CAR construct will optimize the killing of AML cells while minimizing HSC eradication. This novel second-generation CAR has the potential to greatly impact the treatment of AML patients in the future.